Loss-of-Function Mutation in the Dioxygenase-Encoding FTO Causes Severe Growth Retardation and Multiple Malformations Sarah Boissel, Orit Reish, Karine Proulx, Hiroko Kawagoe-Takaki, Barbara Sedgwick, Giles Yeo, David Meyre, Christelle Golzio, Florence Molinari, Noman Kadhom, et al.

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Sarah Boissel, Orit Reish, Karine Proulx, Hiroko Kawagoe-Takaki, Barbara Sedgwick, et al.. Loss-of- Function Mutation in the Dioxygenase-Encoding FTO Gene Causes Severe Growth Retardation and Multiple Malformations. American Journal of Genetics, Elsevier (Cell Press), 2009, 85 (1), pp.106-111. ￿10.1016/j.ajhg.2009.06.002￿. ￿hal-02044723￿

HAL Id: hal-02044723 https://hal.archives-ouvertes.fr/hal-02044723 Submitted on 21 Feb 2019

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Sarah Boissel,1,7 Orit Reish,2,7 Karine Proulx,3,7 Hiroko Kawagoe-Takaki,4 Barbara Sedgwick,4 Giles S.H. Yeo,3 David Meyre,5 Christelle Golzio,1 Florence Molinari,1 Noman Kadhom,1 Heather C. Etchevers,1 Vladimir Saudek,3 I. ,3 Philippe Froguel,5,6 ,4 Stephen O’Rahilly,3 Arnold Munnich,1 and Laurence Colleaux1,*

FTO is a nuclear belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first of the FTO gene have been associated with , the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.

Fto was originally identified in mice as one of the en- by oxidative demethylation.10–14 Fto-deficient mice have coded by the 1.6 Mb deletion causing the fused toes (Ft) been recently described.15 Homozygous mice are mutant, an autosomal-dominant mouse mutation gener- viable but postnatal death occurs frequently and loss of ated by a transgene integration into region D of mouse Fto leads to postnatal growth retardation, significant Chr 8.1 In addition to Fto, the deleted segment also reduction in adipose tissue, and lean body mass. contains three members of the Iroquois gene family (Irx3, We ascertained a large Palestinian Arab consanguineous Irx5, and Irx6, also known as the IrxB complex) as well as multiplex family in which nine affected individuals pre- the Fts/Aktip and Ftm/Rpgrip1l genes. Mice heterozygous sented with a previously unreported polymalformation for the Ft mutation are characterized by partial syndactyly syndrome (Figure 1A). The study was approved by the of forelimbs and thymic hyperplasia.1 Ft/Ft embryos die in supreme Israeli Helsinki Review Board (33/07-08) and midgestation and present many abnormalities including informed consent was obtained from all family members. limb polydactyly and distal truncations, major brain and All affected individuals had postnatal growth retardation, heart defects, delay or absence of neural tube closure, microcephaly, severe psychomotor delay, functional brain and facial structure hypoplasia.2,3 deficits, and characteristic facial dysmorphism. In some Interestingly, a -wide search for type 2 patients, structural brain malformations, cardiac defects, diabetes susceptibility genes identified the strong associa- genital anomalies, and cleft palate were also observed tion of a series of single nucleotide polymorphisms (Table 1). Early lethality resulting from intercurrent infec- (SNPs), in tight linkage disequilibrium with rs9939609 tion or unidentified cause occurred at 1–30 months of age. and located in the first intron of the FTO gene (MIM Extensive biochemical, metabolic, and genetic analyses 610966), with higher (BMI) and obesity failed to diagnose any previously known inherited disor- risk in European cohorts.4,5 This association was replicated ders. Because the pedigree suggested an autosomal-reces- in various European cohorts and several studies have been sive mode of inheritance, we performed a genome-wide performed trying to identify association between the autozygosity screen with the Perkin Elmer Biosystems obesity risk and energy intake/expenditure or eating linkage mapping set (version 1) and identified a unique behavior trait.6–8 However, how FTO variants modulate region of shared homozygosity on 16q12 components of the energy balance remains elusive so far. (Figure S1 available online). Further genotype and haplo- FTO has been recognized as a member of the AlkB-related type analyses reduced the critical region to a 6.5 Mb interval family of non-haem iron- and 2-oxoglutarate-dependent between loci D16S411 and D16S3140 (maximum LOD dioxygenases.9 These non- iron , which score Zmax ¼ 4.16 at q ¼ 0 at the D16S3136 ). No other require Fe2þ as a and 2-oxoglutarate and dioxygen genomic region showed consistent linkage. This genetic as cosubstrates, reverse alkylated DNA and RNA damages interval encompasses 28 genes that were all systematically

1INSERM U781 and De´partement de Ge´ne´tique, Universite´ Paris Descartes, Hoˆpital Necker-Enfants Malades, 75015 Paris, France; 2Department of Medical Genetics, Assaf Harofeh, Medical Center, Zerifin, The Sackler School of Medicine, Tel Aviv University, Tel Aviv 70300, Israel; 3Institute of Metabolic Science, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK; 4Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK; 5CNRS 8090-Institute of Biology, Pasteur Institute, 59019 Lille, France; 6Section of Genomic Medicine, Ham- mersmith Hospital, Imperial College London, London W12 0NN, UK 7These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.ajhg.2009.06.002. ª2009 by The American Society of Human Genetics. All rights reserved.

106 The American Journal of Human Genetics 85, 106–111, July 10, 2009 AB

C

Figure 1. Genetic Analysis of a Family with Ante- and Postnatal Growth Retardation and a Severe Polymalformative Syndrome (A) Pedigree of the family. Filled symbols and slashes indicate affected and deceased infants. (B) Electrophoregrams showing the variation of FTO gene sequence in an affected infant and a healthy control. (C) Part of the multiple sequence alignment of FTO representative orthologs (H.s, Homo sapiens; Ma.m, Macaca mulatta; R.n, Rattus nor- vegicus; Mu.m, Mus musculus; C.f, Canis familiaris; E.c, Equus caballus; Or.a, Ornithorhynchus anatinus; Ov.a, Ovis aries; B.t, Bos taurus; M.d, Monodelphis domestica; G.g, Gallus gallus; D.r, Danio rerio; X.t, Xenopus tropicalis; ABH2 H.s, human ABH2; ABH3 H.s, Human ABH3; AlkB E.c, E. coli AlkB). The conserved residues are highlighted and the highlighted in red is the absolutely conserved arginine involved in the R316Q mutation in patients (Ai, affected infant). Blue strands labeled with roman numerals identify three of the eight b strands that form the conserved double-stranded b-helix of the 2OG-. analyzed at both DNA and/or RNA levels (Table S1). We dren with partially overlapping clinical features were tested identified a homozygous single-nucleotide variation at but no mutation in the FTO gene was identified, strength- cDNA position 947 (c.947G/A) within the FTO gene ening the idea that the new syndrome described here is (Figure 1B). The c.947G/A transition predicts a p.R316Q a very rare condition. substitution. Notably, this amino acid residue is absolutely FTO belongs to the AlkB-related and was conserved across all known FTO paralogs and AlkB ortho- shown to localize in the nucleus.9 Immunofluorescence logs (Figure 1C) and is involved in 2-oxoglutarate coordina- experiments performed on patient and control fibroblasts tion by forming stabilizing salt bridges with the carboxyl- demonstrated that the mutation did not affect its nuclear ates of this cosubstrate.9 This variant cosegregated with localization (data not shown). Murine and human Fto the disease and was not found in 730 control , have been shown to demethylate 3-methylthymine and including 378 chromosomes from individuals of Palesti- 3-methyluracil residues in single-stranded DNA and RNA nian Arab origin. We subsequently undertook to estimate in vitro, albeit with a relatively low efficiency. Fto was less the frequency of FTO sequence variants within the general active on the more common forms of methylated DNA base population. FTO coding and intron- boundaries damage, 1-methyladenine and 3-methylcytosine.9–14,16 sequence was determined in 1492 controls of European Moreover, the recombinant murine Fto protein mutated descent without overt disease or syndromic features. No in the residue corresponding to human R316 amino acid nonsense variant was identified and the prevalence of displays a much reduced or absent DNA demethylation missense variants was 0.87%. In addition, neither homozy- activity. To investigate the functional consequences of the gous missense mutation nor composite heterozygous muta- R316Q mutation, we tested the catalytic activity of the tions were detected in this large series of control subjects wild-type and mutant R316Q FTO in vitro. Two (data not shown), further supporting the hypothesis that different assays were used: the first followed the conversion the R316Q mutation is the disease-causing defect. To esti- of the cosubstrate 2-oxoglutarate to succinate and the mate the prevalence of FTO mutations, 27 unrelated chil- second monitored the ability of FTO to demethylate

The American Journal of Human Genetics 85, 106–111, July 10, 2009 107 3-methylthymine in DNA. Most 2-oxoglutarate- and Fe2þ- Table 1. Major Clinical Features in Affected Individuals with c.947G/A Mutation in FTO dependent dioxygenases slowly catalyze the conversion of 2-oxoglutarate to succinate even in the absence of their Number of Patients Presenting 16 the Symptom/Number prime substrate, and this uncoupled reaction may be Clinical Features of Patients Examined stimulated by substrates or their analogs. We optimized Survival conditions to assay the ability of human recombinant FTO protein to convert 14C-2-oxoglutarate to 14C-succinate Death before 3 years of age 8/8 and found that this activity was stimulated 6- to 8- fold by Build free 3-methylthymidine (Figure 2A). Other Intrauterine growth retardation 3/7 such as 1-methyldeoxyadenosine and thymidine or the

Failure to thrive (severe) 8/8 base 3-methylthymine did not stimulate the activity (data not shown). Interestingly, the R316Q FTO mutant protein CNS had no detectable ability to catalyze the uncoupled reaction Developmental delay (severe) 8/8 and also was not stimulated by 3-methylthymidine 14 Postnatal microcephaly (severe) 8/8 (Figure 2A). The ability of FTO to demethylate C-labeled 3-methylthymine in single-stranded poly(dT) substrate Hypertonicity 6/6 was assayed by measuring the release of 14C-formaldehyde. Hydrocephalus 4/8 The optimum pH for the demethylation of 3-methylthy- Lissencephaly 3/8 mine in DNA by AlkB, ABH2 (MIM 610602), and ABH3 (MIM 610603) is 6 to 6.5 and this is also the case for human Seizures 3/8 FTO. Wild-type FTO11,16,17 actively but slowly demethy- Dandy walker malformation 2/8 lated this substrate whereas the mutant R316Q protein Brain atrophy 1/8 was inactive in the assay (Figure 2B). The defective activity

Heart of FTO, as observed in both assays, is most likely due to the inability of the mutant protein to interact with the Ventricular septal defect, 6/8 atrio ventricular defect, cosubstrate 2-oxoglutarate. atent ductus arteriosus To gain insight into the function of the FTO gene product

Hypertrophic cardiomopathy 4/8 and the pathophysiology of the disease, we investigated the expression of FTO in human embryos and adult tissues. Dysmorphism RT-PCR showed an ubiquitous expression in all fetal and Ante verted nostrils 7/7 adult tissues tested, as has been observed in mice (data not 18 Thin vermilion 7/7 shown). Human embryo sections were hybridized in situ at stages Carnegie (C)15, 33–36 days postfertilization (dpf) Prominent alveolar ridge 6/6 and C18 (44–46 dpf) with antisense probes against FTO Retrognathia 7/7 and sense probes as negative controls. We observed a nearly Coarse face 7/7 ubiquitous expression of FTO, with higher expression in the

Protruding tongue 3/7 central nervous system and the liver. In the heart, the mitral and semilunar valves and the wall of the pulmonary trunk Other stained as did the ventricular myocardium, and a strong Short neck 7/7 and uniform expression was observed in the developing

Cutis marmorata 7/7 pituitary and the frontonasal and mandibular mesenchyme (Figure S2). It is worth noting that this wide spatiotemporal Drumstick fingers 6/6 pattern of expression is consistent with the broad spectrum Brachydactyly 6/6 of clinical manifestations of the disease (Table 1). Toenail hypoplasia 6/6 Interestingly, the cultured skin fibroblasts of patient III.15 displayed an altered morphology (including hyper- Skull asymmetry 6/6 trophy), an increased number of vacuoles and cellular Neuro sensory deafness 5/5 debris (Figure 3A), a reduced life span, and decreased prolif- Weak cry 4/6 erative abilities when compared to controls (Figure 3B).

Umbilical hernia 4/6 Cellular expression of the senescence-associated b-galacto- sidase (SA-b-Gal) is thought to be a reliable indicator of Hypertrophy of labia/genital 4/7 ambiguity/undescending testis the switch mechanism that occurs when cells enter the senescent .19 Notably, a significantly elevated Cleft palate/Bifid uvula 3/6 number of SA-b-Gal-positive cells, increasing with passage Optic disk abnormality 3/7 numbers, was found in patient cultured cells compared to Medical information was available from 8/9 patients. passage-matched control fibroblasts (Figure 3C). Although further analyses on skin fibroblasts of additional patients

108 The American Journal of Human Genetics 85, 106–111, July 10, 2009 AB Figure 2. Biochemical Analyses of the Wild-Type and Mutant FTO Proteins (A) Purified FTO was added to 10 ml reaction mixture containing 50 mM HEPES.KOH (pH 7), 50 mg/ml BSA, 4 mM ascorbate, 75 mM Fe(NH4)2(SO4)2, and 20 mM [5-14C]-2-oxoglutarate (30 mCi/mmol from Moravek Biochemicals) and incubated at 37C for various times. To measure stimulation of this activity by 3-methylthymidine, 1 mM 3-methylthymidine (Moravek Biochemicals) was included in the assay mix. The reaction was stopped by adding 5 ml stop solution containing 20 mM succinate, 20 mM 2-oxo- glutarate followed by 5 ml 160 mM dinitrophenylhy- drazine, which precipitates 2-oxoglutarate. This mix was incubated at room temperature for 30 min. An additional 10 ml 1M 2-oxoglutarate was added and incubated for a further 30 min. The precipitate was removed by centrifugation. Clear supernatant (10 ml) was scintillation counted to monitor the 14C-succinate generated. Time course of activity of 1.5 mM FTO: open and closed squares, wild-type FTO protein; open and closed triangles, mutant R316Q FTO protein. Open symbols and dotted line: without 3-methylthymidine; closed symbols and solid line: with 3-methylthymidine (1 mM). (B) A DNA substrate containing 14C-3-methylthymine was prepared by treating poly(dT) with [14C]-methyl iodide (54 Ci/mmole, Amer- sham Biosciences) as previously described11 and had a specific activity of 1580 cpm/mg poly(dT). FTO was added to the 14C-methylated poly(dT) substrate (1000 cpm) in a 100 ml reaction mix containing 50 mM MES-HCl (pH 6), 75 mM Fe(NH4)2(SO4)2, 100 mM 2-oxoglu- tarate, 2 mM ascorbate, 10 mg/ml bovine serum albumin and incubated at 20C for various times. All assays were performed in triplicate. To stop the reaction, EDTA was added to a final concentration of 10 mM. The polynucleotide substrate was then ethanol precipitated in the presence of carrier calf thymus DNA. Two-thirds of the ethanol-soluble radioactive material was monitored by scintillation counting. Equal volumes of the protein preparations were assayed. Standard error of the mean is shown for each time point. Closed square, 2 mM wild-type FTO protein; closed triangle, 2 mM mutant R316Q FTO protein; open circle, ‘‘mock prep’’ prepared in the absence of recombi- nant FTO expression. Equal volumes of the protein preparations were also examined by SDS-10% PAGE. are necessary to validate these findings, taken together nervous and cardiovascular systems. There are several these data could yet suggest that the defective FTO activity possible reasons for the different findings in our patients may cause premature senescence-like phenotype. versus the Fto/ mouse. First, major phenotypic differ- Although repair of alkylated nucleic acid by oxidative ences between rodent models and harboring demethylation is crucial to maintain genome integrity, no identical mutations are well described. For example, matu- AlkB-related protein mutations have been hitherto identi- rity-onset diabetes of the young subtype 3 (MODY3 [MIM fied in human. The results reported here provide therefore 600496]) is caused by heterozygous mutations in the tran- the first example of a human disorder related to the defect scription factor hepatocyte nuclear factor (HNF)-1a (MIM of an AlkB-related protein. The Escherichia coli AlkB protein 142410).24 By contrast, mice with heterozygous mutations catalyzes the oxidative demethylation of 1-methyladenine, in Hnf-1a gene are phenotypically normal.25 Second, it is 3-methylcytosine, and 3-methylthymine in DNA and RNA. possible that the presence, in the nucleus, of a mutant Eight mammalian orthologs of AlkB have been previously catalytically inactive FTO might have biochemical conse- identified (ABH1 to 8),20 and FTO is the ninth member quences over and above that seen with complete deficiency of this family. Of this family, only ABH2 and ABH3 have of Fto. These could include toxic ‘‘gain-of-function’’ effects been demonstrated to be highly active in repairing methyl- or dominant-negative interference with other related ation damage to DNA.13,14 In comparison, ABH1 (MIM family members. Finally, it is formally possible that the 605345) has a modest activity on 3-methylcytosine in phenotype seen in the human subjects is due to a combina- single-stranded DNA or RNA21 whereas FTO has a similar tion of the mutant FTO and a second, as yet undetected, low activity on 3-methylthymine in these substrates.9,16 mutation in the 6 Mb critical region. The fact that no muta- Mice lacking either Abh2 or Abh3 have no overt phenotype tions were detected on sequencing of the coding regions but the Abh2/ mice accumulate significant levels of and splice junctions of all 28 known and putative genes in 1-methyladenine in their genomes.22 Notably, Abh1/ that linked region makes the chances of such a second mice are viable but have intrauterine growth retardation mutation very small. and defects in placental differentiation.23 A cluster of variants located within the first intron of FTO Surprisingly, although homozygous Fto/ mice have has recently been strongly and reproducibly associated with postnatal growth retardation, significant reduction in human adiposity.4,5 Yet, whether these variants influence adipose tissue, and lean body mass, they are not, however, the risk of obesity directly, via the altered expression of reported to have any developmental abnormalities in the FTO or through other mechanisms, remains questionable. central nervous or cardiovascular systems.15 Thus, humans In this regard, it is worth noting that homozygosity for homozygous for a catalytically inactive FTO and Fto null the R316Q FTO mutation caused severe growth retardation. mice share the growth retardation phenotype but both Detailed anthropometric measurements are not available mutations differ in the impact on development of central on unaffected family members but, noticeably, none of

The American Journal of Human Genetics 85, 106–111, July 10, 2009 109 A Figure 3. Cell Morphology and Proliferative Abili- ties of Cultured Skin Fibroblasts Harboring the R316Q FTO Mutation (A) Fibroblast morphology. The arrow shows altered cell morphology and enlarged cell size of a patient fibroblast. (B) Fibroblast growth curves. Fibroblasts were seeded at a density of 5000 cells/well in 12-well dishes. The number of cells per dish was determined with a CASY cell counter on days 2, 5, 7, 9, and 11 after seeding. Filled and open symbols correspond to patient and control fibroblasts, respectively. Data B correspond to the mean of three replicates. Standard error of the mean is shown for each point. (C) Senescence-associated b-galactosidase assay. Fibroblasts were seeded on 6-well dishes at a density of 130,000 cells/well. Senescent fibroblasts were stained with the Senescence b-Galactosidase Staining Kit (Cell Signaling Technology). Percentages of b-galactosidase-positive cells for passages 7, 9, and 13 were calculated on the basis of approximately 100 cells. Data correspond to the mean of three replicates. Standard error of the mean is shown for each point.

C ciently, suggesting that this is not its true phys- iological substrate, (3) 3-methylthymine is a very rare lesion in vivo, and (4) defective repair of rare methylated bases in DNA can hardly be related to the striking increase in energy expen- diture observed in Fto-deficient mice. Taken together, this suggests that FTO roles in the nucleus are presumably not restricted to DNA repair. Because of its association with obesity, FTO is currently the subject of intense interest. The finding of obesity resistance in Fto-deficient mice has led to the suggestion that interference the parents were clinically obese. Given the results from the with Fto enzymatic function might be a novel and inter- Fto-deficient mice, it might be hypothesized that humans esting antiobesity therapeutic strategy. The discovery that heterozygous for a loss-of-function mutation in FTO might humans homozygous for an enzymatically inactive mutant actually be relatively resistant to becoming obese. Future form of FTO have multiple congenital abnormalities suggests studies of the body composition of carrier and that any program of research exploring the therapeutic noncarrier relatives will be required to clarify the relation- utility of FTO inhibitors should incorporate a particularly ship between FTO variant heterozygosity and adiposity. careful assessment of teratogenicity and other toxic effects. The molecular mechanisms whereby the mutant FTO leads to the severe phenotype observed in our patients Supplemental Data remain unknown. The fact that (1) FTO is a member of the Alk-B-related family of dioxygenases, (2) at least two Supplemental data include two figures and one table and can be of the mammalian members of this family have established found with this article online at http://www.ajhg.org/. roles in DNA repair, and (3) FTO can demethylate 3-methyl- thymine in the context of DNA might suggest that FTO Acknowledgments plays a role in the maintenance of genome integrity. If Weare grateful to the patients for their participation in the study. We that were the case, one could easily understand how FTO acknowledge Jean Philippe Jais for LOD score calculation, Carron defect relates to developmental malformations and the Sher for participation to clinical evaluation, Uli Ru¨ther for kindly accelerated senescence observed in cultured fibroblasts providing FTO , Ce´line Cluzeau for help with western derived from the patients. However, it should also be blotting, Peter Robins for purification of FTO protein, and Marcella pointed out that (1) several members of the mammalian Ma and Debbie Lyon for additional technical assistance. This study Alk-B-related family have no established role in DNA repair, was supported by the Centre National de la Recherche Scientifique, (2) FTO demethylates 3-methylthymine slowly and ineffi- the Agence Nationale de la Recherche, the Re´gion Ile-de-France, EU

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